Oligonucleotide modulation of cell adhesion

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to treatment through modulation of the synthesis or metabolism of intercellular adhesion molecules. In accordance with preferred embodiments, oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1. The oligonucleotide comprises nucleotide units sufficient in identity and number to effect said specific hybridization. In other preferred embodiments, the oligonucleotides are specifically hybridizable with a transcription initiation site, a translation initiation site, 5&#39;-untranslated sequences, 3&#39;-untranslated sequences, and intervening sequences. Methods of treating animals suffering from disease amenable to therapeutic intervention by modulating cell adhesion proteins with an oligonucleotide specifically hybridizable with RNA or DNA corresponding to one of the foregoing proteins are disclosed. Methods for treatment of diseases responding to modulation cell adhesion molecules are disclosed.

This application is a continuation of Ser. No: 08/440,740 filed May 12,1995, now U.S. Pat. No. 5,843,738, which is a is a continuation-in-partof application Ser. No. 08/063,167, filed May 17, 1993, now U.S. Pat.No. 5,514,788, which is a continuation of application Ser. No.07/969,151, filed Feb. 10, 1993, now abandoned, which is acontinuation-in-part of application Ser. No. 08/007,997, filed Jan. 21,1993, now U.S. Pat. No. 5,591,623, which is a continuation-in-part ofapplication Ser. No. 07/939,855, filed Sep. 2, 1992, now abandoned,which is a continuation-in-part of application Ser. No. 07/567,286 filedAug. 14, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to diagnostics, research reagents and therapiesfor disease states which respond to modulation of the synthesis ormetabolism of cell adhesion molecules. In particular, this inventionrelates to antisense oligonucleotide interactions with certain messengerribonucleic acids (mRNAs) or DNAs involved in the synthesis of proteinsthat regulate adhesion of white blood cells to other white blood cellsand to other cell types. Antisense oligonucleotides designed tohybridize to the mRNA encoding intercellular adhesion molecule-1(ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1, also knownas E-selectin), and vascular cell adhesion molecule-1 (VCAM-1) areprovided. These oligonucleotides have been found to lead to themodulation of the activity of the RNA or DNA, and thus to the modulationof the synthesis and metabolism of specific cell adhesion molecules.Palliation and therapeutic effect result.

BACKGROUND OF THE INVENTION

Inflammation is a localized protective response elicited by tissues inresponse to injury, infection, or tissue destruction resulting in thedestruction of the infectious or injurious agent and isolation of theinjured tissue. A typical inflammatory response proceeds as follows:recognition of an antigen as foreign or recognition of tissue damage,synthesis and release of soluble inflammatory mediators, recruitment ofinflammatory cells to the site of infection or tissue damage,destruction and removal of the invading organism or damaged tissue, anddeactivation of the system once the invading organism or damage has beenresolved. In many human diseases with an inflammatory component, thenormal, homeostatic mechanisms which attenuate the inflammatoryresponses are defective, resulting in damage and destruction of normaltissue.

Cell-cell interactions are involved in the activation of the immuneresponse at each of the stages described above. One of the earliestdetectable events in a normal inflammatory response is adhesion ofleukocytes to the vascular endothelium, followed by migration ofleukocytes out of the vasculature to the site of infection or injury.The adhesion of these leukocytes, or white blood cells, to vascularendothelium is an obligate step in the migration out of the vasculature.Harlan, J. M., Blood 1985, 65, 513-525. In general, the firstinflammatory cells to appear at the site of inflammation are neutrophilsfollowed by monocytes, and lymphocytes. Cell--cell interactions are alsocritical for propagation of both B-lymphocytes and T-lymphocytesresulting in enhanced humoral and cellular immune responses,respectively.

The adhesion of white blood cells to vascular endothelium and other celltypes is mediated by interactions between specific proteins, termed"adhesion molecules," located on the plasma membrane of both white bloodcells and vascular endothelium. The interaction between adhesionmolecules is similar to classical receptor ligand interactions with theexception that the ligand is fixed to the surface of a cell instead ofbeing soluble. The identification of patients with a genetic defect inleukocyte adhesion has enabled investigators to identify a family ofproteins responsible for adherence of white blood cells. Leukocyteadhesion deficiency (LAD) is a rare autosomal trait characterized byrecurrent bacterial infections and impaired pus formation and woundhealing. The defect was shown to occur in the common B-subunit of threeheterodimeric glycoproteins, Mac-1, LFA-1, and p150,95, normallyexpressed on the outer cell membrane of white blood cells. Anderson andSpringer, Ann. Rev. Med. 1987, 38, 175-194. Patients suffering from LADexhibit a defect in a wide spectrum of adherence-dependent functions ofgranulocytes, monocytes, and lymphocytes. Three ligands for LFA-1 havebeen identified, intercellular adhesion molecules 1, 2 and 3 (ICAM-1,ICAM-2 and ICAM-3). Both Mac-1 and p150,95 bind complement fragment C3biand perhaps other unidentified ligands. Mac-1 also binds ICAM-1.

Other adhesion molecules have been identified which are involved in theadherence of white blood cells to vascular endothelium and subsequentmigration out of the vasculature. These include endothelial leukocyteadhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1)and granule membrane protein-140 (GMP-140) and their respectivereceptors. The adherence of white blood cells to vascular endotheliumappears to be mediated in part if not in toto by the five cell adhesionmolecules ICAM-1, ICAM-2, ELAM-1, VCAM-1 and GMP-140. Dustin andSpringer, J. Cell Biol. 1987, 107, 321-331. Expression on the cellsurface of ICAM-1, ELAM-1, VCAM-1 and GMP-140 adhesion molecules isinduced by inflammatory stimuli. In contrast, expression of ICAM-2appears to be constitutive and not sensitive to induction by cytokines.In general, GMP-140 is induced by autocoids such as histamine,leukotriene B₄, platelet activating factor, and thrombin. Maximalexpression on endothelial cells is observed 30 minutes to 1 hour afterstimulation, and returns to baseline within 2 to 3 hours. The expressionof ELAN-1 and VCAM-1 on endothelial cells is induced by cytokines suchas interleukin-1β and tumor necrosis factor, but not gamma-interferon.Maximal expression of ELAM-1 on the surface of endothelial cells occurs4 to 6 hours after stimulation, and returns to baseline by 16 hours.ELAM-1 expression is dependent on new mRNA and protein synthesis.Elevated VCAM-1 expression is detectable 2 hours following treatmentwith tumor necrosis factor, is maximal 8 hours following stimulation,and remains elevated for at least 48 hours following stimulation. Riceand Bevilacqua, Science 1989, 246, 1303-1306. ICAM-1 expression onendothelial cells is induced by cytokines interleukin-1 tumor necrosisfactor and gamma-interferon. Maximal expression of ICAM-1 follows thatof ELAM-1 occurring 8 to 10 hours after stimulation and remains elevatedfor at least 48 hours.

GMP-140 and ELAM-1 are primarily involved in the adhesion of neutrophilsto vascular endothelial cells. VCAM-1 primarily binds T and Blymphocytes. In addition, VCAM-1 may play a role in the metastasis ofmelanoma, and possibly other cancers. ICAM-1 plays a role in adhesion ofneutrophils to vascular endothelium, as well as adhesion of monocytesand lymphocytes to vascular endothelium, tissue fibroblasts andepidermal keratinocytes. ICAM-1 also plays a role in T-cell recognitionof antigen presenting cell, lysis of target cells by natural killercells, lymphocyte activation and proliferation, and maturation of Tcells in the thymus. In addition, recent data have demonstrated thatICAM-1 is the cellular receptor for the major serotype of rhinovirus,which account for greater than 50% of common colds. Staunton et al.,Cell 1989, 56, 849-853; Greve et al., Cell 1989, 56, 839-847.

Expression of ICAM-1 has been associated with a variety of inflammatoryskin disorders such as allergic contact dermatitis, fixed drug eruption,lichen planus, and psoriasis; Ho et al., J. Am. Acad. Dermatol. 1990,22, 64-68; Griffiths and Nickoloff, Am. J. Pathology 1989, 135,1045-1053; Lisby et al., Br. J. Dermatol. 1989,120, 479-484; Shiohara etal., Arch. Dermatol. 1989, 125, 1371-1376. In addition, ICAM-1expression has been detected in the synovium of patients with rheumatoidarthritis; Hale et al., Arth. Rheum. 1989, 32, 22-30, pancreatic B-cellsin diabetes; Campbell et al., Proc. Natl. Acad. Sci. U.S.A. 1989, 86,4282-4286; thyroid follicular cells in patients with Graves' disease;Weetman et al., J. Endocrinol. 1989, 122, 185-191; and with renal andliver allograft rejection; Faull and Russ, Transplantation 1989, 48,226-230; Adams et al., Lancet 1989, 1122-1125.

It is has been hoped that inhibitors of ICAM-1, VCAM-1 and ELAN-1expression would provide a novel therapeutic class of anti-inflammatoryagents with activity towards a variety of inflammatory diseases ordiseases with an inflammatory component such as asthma, rheumatoidarthritis, allograft rejections, inflammatory bowel disease, variousdermatological conditions, and psoriasis. In addition, inhibitors ofICAM-1, VCAM-1, and ELAM-1 may also be effective in the treatment ofcolds due to rhinovirus infection, AIDS, Kaposi's sarcoma and somecancers and their metastasis. To date, there are no known therapeuticagents which effectively prevent the expression of the cellular adhesionmolecules ELAM-1, VCAM-1 and ICAM-1. The use of neutralizing monoclonalantibodies against ICAM-1 in animal models provide evidence that suchinhibitors if identified would have therapeutic benefit for asthma;Wegner et al., Science 1990, 247, 456-459, renal allografts; Cosimi etal., J. Immunol. 1990, 144, 4604-4612, and cardiac allografts; Isobe etal., Science 1992, 255, 1125-1127. The use of a soluble form of ICAM-1molecule was also effective in preventing rhinovirus infection of cellsin culture. Marlin et al., Nature 1990, 344, 70-72.

Current agents which affect intercellular adhesion molecules includesynthetic peptides, monoclonal antibodies, and soluble forms of theadhesion molecules. To date, synthetic peptides which block theinteractions with VCAM-1 or ELAM-1 have not been identified. Monoclonalantibodies may prove to be useful for the treatment of acuteinflammatory response due to expression of ICAM-1, VCAM-1 and ELAM-1.However, with chronic treatment, the host animal develops antibodiesagainst the monoclonal antibodies thereby limiting their usefulness. Inaddition, monoclonal antibodies are large proteins which may havedifficulty in gaining access to the inflammatory site. Soluble forms ofthe cell adhesion molecules suffer from many of the same limitations asmonoclonal antibodies in addition to the expense of their production andtheir low binding affinity. Thus, there is a long felt need formolecules which effectively inhibit intercellular adhesion molecules.Antisense oligonucleotides avoid many of the pitfalls of current agentsused to block the effects of ICAM-1, VCAM-1 and ELAM-1.

PCT/US90/02357 (Hession et al.) discloses DNA sequences encodingEndothelial Adhesion Molecules (ELAMs), including ELAM-1 and VCAM-1 andVCAM-1b. A number of uses for these DNA sequences are provided,including (1) production of monoclonal antibody preparations that arereactive for these molecules which may be used as therapeutic agents toinhibit leukocyte binding to endothelial cells; (2) production of ELAMpeptides to bind to the ELAM ligand on leukocytes which, in turn, maybind to ELAM on endothelial cells, inhibiting leukocyte binding toendothelial cells; (3) use of molecules binding to ELAMS (such asanti-ELAM antibodies, or markers such as the ligand or fragments of it)to detect inflammation; (4) use of ELAM and ELAM ligand DNA sequences toproduce nucleic acid molecules that intervene in ELAM or ELAM ligandexpression at the translational level using antisense nucleic acid andribozymes to block translation of a specific MRNA either by masking MRNAwith antisense nucleic acid or cleaving it with a ribozyme. It isdisclosed that coding regions are the targets of choice. For VCAM-1, AUGis believed to be most likely; a 15-mer hybridizing to the AUG site isspecifically disclosed in Example 17.

OBJECTS OF THE INVENTION

It is a principle object of the invention to provide therapies fordiseases with an immunological component, allografts, cancers andmetastasis, inflammatory bowel disease, psoriasis and other skindiseases, colds, and AIDS through perturbation in the synthesis andexpression of inflammatory cell adhesion molecules.

It is a further object of the invention to provide antisenseoligonucleotides which are capable of inhibiting the function of nucleicacids encoding intercellular adhesion proteins.

Yet another object is to provide means for diagnosis of dysfunctions ofintercellular adhesion.

These and other objects of this invention will become apparent from areview of the instant specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the mRNA sequence of human intercellular adhesion molecule-1(ICAM-1).

FIG. 2 is the mRNA sequence of human endothelial leukocyte adhesionmolecule-1 (ELAM-1).

FIG. 3 is the mRNA sequence of human vascular cell adhesion molecule-1(VCAM-1).

FIG. 4 is a graphical representation of the induction of ICAM-1expression on the cell surface of various human cell lines byinterleukin-1 and tumor necrosis factor.

FIG. 5 is a graphical representation of the effects of selectedantisense oligonucleotides on ICAM-1 expression on human umbilical veinendothelial cells.

FIGS. 6A and 6B are a graphical representation of the effects of anantisense oligonucleotide on the expression of ICAM-1 in human umbilicalvein endothelial cells stimulated with tumor necrosis factor andinterleukin-1.

FIG. 7 is a graphical representation of the effect of antisenseoligonucleotides on ICAM-1 mediated adhesion of DMSO differentiatedHL-60 cells to control and tumor necrosis factor treated human umbilicalvein endothelial cells.

FIG. 8 is a graphical representation of the effects of selectedantisense oligonucleotides on ICAM-1 expression in A549 human lungcarcinoma cells.

FIG. 9 is a graphical representation of the effects of selectedantisense oligonucleotides on ICAM-1 expression in primary humankeratinocytes.

FIG. 10 is a graphical representation of the relationship betweenoligonucleotide chain length, Tm and effect on inhibition of ICAM-1expression.

FIG. 11 is a graphical representation of the effect of selectedantisense oligonucleotides on ICAM-1 mediated adhesion of DMSOdifferentiated HL-60 cells to control and tumor necrosis factor treatedhuman umbilical vein endothelial cells.

FIG. 12 is a graphical representation of the effects of selectedantisense oligonucleotides on ELAM-1 expression on tumor necrosisfactor-treated human umbilical vein endothelial cells.

FIG. 13 is a graphical representation of the human ELAM-1 mRNA showingtarget sites of antisense oligonucleotides.

FIG. 14 is a graphical representation of the human VCAM-1 mRNA showingtarget sites of antisense oligonucleotides.

FIG. 15 is a line graph showing inhibition of ICAM-1 expression in C8161human melanoma cells following treatment with antisense oligonucleotidescomplementary to ICAM-1.

FIG. 16 is a bar graph showing the effect of ISIS 3082 on dextransulfate (DSS)-induced inflammatory bowel disease.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are providedwhich specifically hybridize with nucleic acids encoding intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)and endothelial leukocyte adhesion molecule-1 (ELAM-1). Theoligonucleotides are designed to bind either directly to mRNA or to aselected DNA portion forming a triple stranded structure, therebymodulating the amount of mRNA made from the gene. This relationship iscommonly denoted as "antisense."

Oligonucleotides are commonly used as research reagents and diagnostics.For example, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes, forexample to distinguish between the functions of various members of abiological pathway. This specific inhibitory effect has, therefore, beenharnessed for research use. This specificity and sensitivity is alsoharnessed by those of skill in the art for diagnostic uses.

It is preferred to target specific genes for antisense attack."Targeting" an oligonucleotide to the associated ribonucleotides, in thecontext of this invention, is a multistep process. The process usuallybegins with identifying a nucleic acid sequence whose function is to bemodulated. This may be, as examples, a cellular gene (or mRNA made fromthe gene) whose expression is associated with a particular diseasestate, or a foreign nucleic acid from an infectious agent. In thepresent invention, the target is a cellular gene associated with aparticular disease state. The targeting process also includesdetermination of a site or sites within this region for theoligonucleotide interaction to occur such that the desired effect,either detection of or modulation of expression of the protein, willresult. Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

It has been discovered that the genes coding for ICAM-1, VCAM-1 andELAM-1 are particularly useful for this approach. Inhibition of ICAM-1,VCAM-1 and ELAM-1 expression is expected to be useful for the treatmentof inflammatory diseases, diseases with an inflammatory component,allograft rejection, psoriasis and other skin diseases, inflammatorybowel disease, cancers and their metastasis, and viral infections.

Methods of modulating cell adhesion comprising contacting the animalwith an oligonucleotide hybridizable with nucleic acids encoding aprotein capable of modulating cell adhesion are provided.Oligonucleotides hybridizable with an RNA or DNA encoding ICAM-1, VCAM-1and ELAM-1 are preferred.

The present invention is also useful in diagnostics and in research.Since the oligonucleotides of this invention hybridize to ICAM-1, ELAM-1or VCAM-1, sandwich and other assays can easily be constructed toexploit this fact. Provision of means for detecting hybridization of anoligonucleotide with one of these intercellular adhesion moleculespresent in a sample suspected of containing it can routinely beaccomplished. Such provision may include enzyme conjugation,radiolabelling or any other suitable detection system. A number ofassays may be formulated employing the present invention, which assayswill commonly comprise contacting a tissue sample with a detectablylabeled oligonucleotide of the present invention under conditionsselected to permit hybridization and measuring such hybridization bydetection of the label.

For example, radiolabeled oligonucleotides can be prepared by ³² Plabeling at the 5' end with polynucleotide kinase. Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, Volume 2, pg. 10.59. Radiolabeled oligonucleotides are thencontacted with tissue or cell samples suspected of containing anintercellular adhesion molecule and the sample is washed to removeunbound oligonucleotide. Radioactivity remaining in the sample indicatesbound oligonucleotide (which in turn indicates the presence of anintercellular adhesion molecule) and can be quantitated using ascintillation counter or other routine means. Expression of theseproteins can then be detected.

Radiolabeled oligonucleotides of the present invention can also be usedto perform autoradiography of tissues to determine the localization,distribution and quantitation of intercellular adhesion molecules forresearch, diagnostic or therapeutic purposes. In such studies, tissuesections are treated with radiolabeled oligonucleotide and washed asdescribed above, then exposed to photographic emulsion according toroutine autoradiography procedures. The emulsion, when developed, yieldsan image of silver grains over the regions expressing a intercellularadhesion molecule. Quantitation of the silver grains permits expressionof these molecules to be detected and permits targeting ofoligonucleotides to these areas.

Analogous assays for fluorescent detection of expression ofintercellular adhesion molecules can be developed using oligonucleotidesof the present invention which are conjugated with fluorescein or otherfluorescent tag instead of radiolabeling. Such conjugations areroutinely accomplished during solid phase synthesis using fluorescentlylabeled amidites or CPG (e.g., fluorescein-labeled amidites and CPGavailable from Glen Research, Sterling Va.).

Each of these assay formats is known in the art. One of skill couldeasily adapt these known assays for detection of expression ofintercellular adhesion molecules in accordance with the teachings of theinvention providing a novel and useful means to detect expression ofthese molecules. Antisense oligonucleotide inhibition of the expressionof intercellular adhesion molecules in vitro is useful as a means todetermine a proper course of therapeutic treatment. For example, beforea patient is treated with an oligonucleotide composition of the presentinvention, cells, tissues or a bodily fluid from the patient can betreated with the oligonucleotide and inhibition of expression ofintercellular adhesion molecules can be assayed. Effective in vitroinhibition of the expression of molecules in the sample indicates thatthe expression will also be modulated in vivo by this treatment.

Kits for detecting the presence or absence of intercellular adhesionmolecules may also be prepared. Such kits include an oligonucleotidetargeted to ICAM-1, ELAM-1 or VCAM-1.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Antisense oligonucleotides hold great promise as therapeutic agents forthe treatment of many human diseases. Oligonucleotides specifically bindto the complementary sequence of either pre-mRNA or mature mRNA, asdefined by Watson-Crick base pairing, inhibiting the flow of geneticinformation from DNA to protein. The properties of antisenseoligonucleotides which make them specific for their target sequence alsomake them extraordinarily versatile. Because antisense oligonucleotidesare long chains of four monomeric units they may be readily synthesizedfor any target RNA sequence. Numerous recent studies have documented theutility of antisense oligonucleotides as biochemical tools for studyingtarget proteins. Rothenberg et al., J. Natl. Cancer Inst. 1989, 81,1539-1544; Zon, G. Pharmaceutical Res. 1988, 5, 539-549). Because ofrecent advances in synthesis of nuclease resistant oligonucleotides,which exhibit enhanced cell uptake, it is now possible to consider theuse of antisense oligonucleotides as a novel form of therapeutics.

Antisense oligonucleotides offer an ideal solution to the problemsencountered in prior art approaches. They can be designed to selectivelyinhibit a given isoenzyme, they inhibit the production of the enzyme,and they avoid non-specific mechanisms such as free radical scavengingor binding to multiple receptors. A complete understanding of enzymemechanisms or receptor-ligand interactions is not needed to designspecific inhibitors.

Description of Targets

The acute infiltration of neutrophils into the site of inflammationappears to be due to increased expression of GMP-140, ELAM-1 and ICAM-1on the surface of endothelial cells. The appearance of lymphocytes andmonocytes during the later stages of an inflammatory reaction appear tobe mediated by VCAM-1 and ICAM-1. ELAM-1 and GMP-140 are transientlyexpressed on vascular endothelial cells, while VCAM-1 and ICAM-1 arechronically expressed.

Human ICAM-1 is encoded by a 3.3-kb mRNA resulting in the synthesis of a55,219 dalton protein (FIG. 1). ICAM-1 is heavily glycosylated throughN-linked glycosylation sites. The mature protein has an apparentmolecular mass of 90 kDa as determined by SDS-polyacrylamide gelelectrophoresis. Staunton et al., Cell 1988, 52, 925-933. ICAM-1 is amember of the immunoglobulin supergene family, containing 5immunoglobulin-like domains at the amino terminus, followed by atransmembrane domain and a cytoplasmic domain. The primary binding sitefor LFA-1 and rhinovirus are found in the first immunoglobulin-likedomain. However, the binding sites appear to be distinct. Staunton etal., Cell 1990, 61, 243-354. Recent electron micrographic studiesdemonstrate that ICAM-1 is a bent rod 18.7 nm in length and 2 to 3 nm indiameter. Staunton et al., Cell 1990, 61, 243-254.

ICAM-1 exhibits a broad tissue and cell distribution, and may be foundon white blood cells, endothelial cells, fibroblast, keratinocytes andother epithelial cells. The expression of ICAM-1 can be regulated onvascular endothelial cells, fibroblasts, keratinocytes, astrocytes andseveral cell lines by treatment with bacterial lipopolysaccharide andcytokines such as interleukin-1, tumor necrosis factor,gamma-interferon, and lymphotoxin. See, e.g., Frohman et al., J.Neuroimmunol. 1989, 23, 117-124. The molecular mechanism for increasedexpression of ICAM-1 following cytokine treatment has not beendetermined.

ELAM-1 is a 115-kDa membrane glycoprotein (FIG. 2) which is a member ofthe selectin family of membrane glycoproteins. Bevilacqua et al.,Science 1989, 243, 1160-1165. The amino terminal region of ELAM-1contains sequences with homologies to members of lectin-like proteins,followed by a domain similar to epidermal growth factor, followed by sixtandem 60-amino acid repeats similar to those found in complementreceptors 1 and 2. These features are also shared by GMP-140 and MEL-14antigen, a lymphocyte homing antigen. ELAM-1 is encoded for by a 3.9-kbmRNA. The 3'-untranslated region of ELAM-1 mRNA contains severalsequence motifs ATTTA which are responsible for the rapid turnover ofcellular mRNA consistent with the transient nature of ELAM-1 expression.

ELAM-1 exhibits a limited cellular distribution in that it has only beenidentified on vascular endothelial cells. Like ICAM-1, ELAM-1 isinducible by a number of cytokines including tumor necrosis factor,interleukin-1 and lymphotoxin and bacterial lipopolysaccharide. Incontrast to ICAM-1, ELAM-1 is not induced by gamma-interferon.Bevilacqua et al., Proc. Natl. Acad. Sci. USA 1987, 84, 9238-9242;Wellicome et al., J. Immunol. 1990, 144, 2558-2565. The kinetics ofELAM-1 mRNA induction and disappearance in human umbilical veinendothelial cells precedes the appearance and disappearance of ELAM-1 onthe cell surface. As with ICAM-1, the molecular mechanism for ELAM-1induction is not known.

VCAM-1 is a 110-kDa membrane glycoprotein encoded by a 3.2-kb mRNA (FIG.3). VCAM-1 appears to be encoded by a single-copy gene which can undergoalternative splicing to yield products with either six or sevenimmunoglobulin domains. Osborn et al., Cell 1989, 59, 1203-1211. Thereceptor for VCAM-1 is proposed to be CD29 (VLA-4) as demonstrated bythe ability of monoclonal antibodies to CD29 to block adherence of Ramoscells to VCAM-1. VCAM-1 is expressed primarily on vascular endothelialcells. Like ICAM-1 and ELAM-1, expression of VCAM-1 on vascularendothelium is regulated by treatment with cytokines. Rice andBevilacqua, Science 1989, 246, 1303-1306; Rice et al., J. Exp. Med.1990, 171, 1369-1374. Increased expression appears to be due toinduction of the mRNA.

For therapeutics, an animal suspected of having a disease which can betreated by decreasing the expression of ICAM-1, VCAM-1 and ELAM-1 istreated by administering oligonucleotides in accordance with thisinvention. Oligonucleotides may be formulated in a pharmaceuticalcomposition, which may include carriers, thickeners, diluents, buffers,preservatives, surface active agents, liposomes or lipid formulationsand the like, in addition to the oligonucleotide. Pharmaceuticalcompositions may also include one or more active ingredients such asantimicrobial agents, anti-inflammatory agents, anesthetics, and thelike, in addition to oligonucleotide.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms orgloves may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, liposomes, diluents and othersuitable additives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until a cureis effected or a diminution of disease state is achieved. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

The present invention employs oligonucleotides for use in antisenseinhibition of the function of RNA and DNA corresponding to proteinscapable of modulating inflammatory cell adhesion. In the context of thisinvention, the term "oligonucleotide" refers to an oligomer or polymerof ribonucleic acid or deoxyribonucleic acid. This term includesoligomers consisting of naturally occurring bases, sugars and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced cellular uptake and increasedstability in the presence of nucleases.

Specific examples of some preferred oligonucleotides envisioned for thisinvention may contain phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages. Mostpreferred are those with CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂, CH₂--O--N(CH₃)--CH₂, CH₂ --N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂backbones (where phosphodiester is O--P--O--CH₂). Also preferred areoligonucleotides having morpholino backbone structures. Summerton, J. E.and Weller, D. D., U.S. Pat. No. 5,034,506. In other preferredembodiments, such as the protein-nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide may be replaced with apolyamide backbone, the bases being bound directly or indirectly to theaza nitrogen atoms of the polyamide backbone. P. E. Nielsen, M. Egholm,R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferredoligonucleotides may contain alkyl and halogen-substituted sugarmoieties comprising one of the following at the 2' position: OH, SH,SCH₃, F, OCN, O(CH₂)_(n) NH₂ or O(CH₂)_(n) CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O--, S--, or N-alkyl; O--, S--, orN-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a conjugate; a reporter group; an intercalator; agroup for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide and other substituents having similar properties.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pencofuranosyl group.

The oligonucleotides in accordance with this invention preferablycomprise from about 3 to about 50 nucleic acid base units. It is morepreferred that such oligonucleotides comprise from about 8 to 25 nucleicacid base units, and still more preferred to have from about 12 to 22nucleic acid base units. As will be appreciated, a nucleic acid baseunit is a base-sugar combination suitably bound to an adjacent nucleicacid base unit through phosphodiester or other bonds.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; however, the actual synthesis of theoligonucleotides are well within the talents of the routineer. It isalso well known to use similar techniques to prepare otheroligonucleotides such as the phosphorothioates and alkylatedderivatives.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA identified by the open reading frames(ORFs) of the DNA from which they are transcribed includes not only theinformation from the ORFs of the DNA, but also associatedribonucleotides which form regions known to such persons as the5'-untranslated region, the 3'- untranslated region, and interveningsequence ribonucleotides. Thus, oligonucleotides may be formulated inaccordance with this invention which are targeted wholly or in part tothese associated ribonucleotides as well as to the informationalribonucleotides. In preferred embodiments, the oligonucleotide isspecifically hybridizable with a transcription initiation site, atranslation initiation site, an intervening sequence and sequences inthe 3'-untranslated region.

In accordance with this invention, the oligonucleotide is specificallyhybridizable with portions of nucleic acids encoding a protein involvedin the adhesion of white blood cells either to other white blood cellsor other cell types. In preferred embodiments, said proteins areintercellular adhesion molecule-1, vascular cell adhesion molecule-1 andendothelial leukocyte adhesion molecule-1. Oligonucleotides comprisingthe corresponding sequence, or part thereof, are useful in theinvention. For example, FIG. 1 is a human intercellular adhesionmolecule-1 mRNA sequence. A preferred sequence segment which may beuseful in whole or in part is:

    ______________________________________                                        5'                3'    SEQ ID NO:                                            ______________________________________                                        TGGGAGCCATAGCGAGGC  1                                                         GAGGAGCTCAGCGTCGACTG                                                                              2                                                         GACACTCAATAAATAGCTGGT                                                                             3                                                         GAGGCTGAGGTGGGAGGA  4                                                         CGATGGGCAGTGGGAAAG  5                                                         GGGCGCGTGATCCTTATAGC                                                                              6                                                         CATAGCGAGGCTGAGGTTGC                                                                              7                                                         CGGGGGCTGCTGGGAGCCAT                                                                              8                                                         TCAGGGAGGCGTGGCTTGTG                                                                              13                                                        CCTGTCCCGGGATAGGTTCA                                                                              14                                                        TTGAGAAAGCTTTATTAACT                                                                              16                                                        CCCCCACCACTTCCCCTCTC.                                                                             15                                                        ______________________________________                                    

FIG. 2 is a human endothelial leukocyte adhesion molecule-1 mRNAsequence. A preferred sequence segment which may be useful in whole orin part is:

    ______________________________________                                        5'                  3'    SEQ ID NO:                                          ______________________________________                                        CAATCATGACTTCAAGAGTTCT                                                                              28                                                      TCACTGCTGCCTCTGTCTCAGG                                                                              73                                                      TGATTCTTTTGAACTTAAAAGGA                                                                             74                                                      TTAAAGGATGTAAGAAGGCT  75                                                      CATAAGCACATTTATTGTC   76                                                      TTTTGGGAAGCAGTTGTTCA  77                                                      AACTGTGAAGCAATCATGACT 78                                                      CCTTGAGTGGTGCATTCAACCT                                                                              79                                                      AATGCTTGCTCACACAGGCATT.                                                                             80                                                      ______________________________________                                    

FIG. 3 is a human vascular cell adhesion molecule-1 mRNA sequence. Apreferred sequence segment which may be useful in whole or in part is:

    ______________________________________                                        5'                  3'    SEQ ID NO:                                          ______________________________________                                        CCAGGCATTTTAAGTTGCTGT 40                                                      CCTGAAGCCAGTGAGGCCCG  41                                                      GATGAGAAAATAGTGGAACCA 42                                                      CTGAGCAAGATATCTAGAT   43                                                      CTACACTTTTGATTTCTGT   44                                                      TTGAACATATCAAGCATTAGCT                                                                              45                                                      TTTACATATGTACAAATTATGT                                                                              46                                                      AATTATCACTTTACTATACAAA                                                                              47                                                      AGGGCTGACCAAGACGGTTGT.                                                                              48                                                      ______________________________________                                    

While the illustrated sequences are believed to be accurate, the presentinvention is directed to the correct sequences, should errors be found.Oligonucleotides useful in the invention comprise one of thesesequences, or part thereof. Thus, it is preferred to employ any of theseoligonucleotides as set forth above or any of the similaroligonucleotides which persons of ordinary skill in the art can preparefrom knowledge of the preferred antisense targets for the modulation ofthe synthesis of inflammatory cell adhesion molecules.

Several preferred embodiments of this invention are exemplified inaccordance with the following nonlimiting examples. The target mRNAspecies for modulation relates to intercellular adhesion molecule-1,endothelial leukocyte adhesion molecule-1, and vascular cell adhesionmolecule-1. Persons of ordinary skill in the art will appreciate thatthe present invention is not so limited, however, and that it isgenerally applicable. The inhibition or modulation of production of theICAM-1 and/or ELAM-1 and/or VCAM-1 are expected to have significanttherapeutic benefits in the treatment of disease. In order to assess theeffectiveness of the compositions, an assay or series of assays isrequired.

The following examples are provided for illustrative purposes only andare not intended to limit the invention.

EXAMPLES Example 1

Expression of ICAM-1, VCAM-1 and ELAM-1 on the surface of cells can bequantitated using specific monoclonal antibodies in an ELISA. Cells aregrown to confluence in 96 well microliter plates. The cells arestimulated with either interleukin-1 or tumor necrosis factor for 4 to 8hours to quantitate ELAM-1 and 8 to 24 hours to quantitate ICAM-1 andVCAM-1. Following the appropriate incubation time with the cytokine, thecells are gently washed three times with a buffered isotonic solutioncontaining calcium and magnesium such as Dulbecco's phosphate bufferedsaline (D-PBS). The cells are then directly fixed on the microtiterplate with 1 to 2% paraformaldehyde diluted in D-PBS for 20 minutes at25° C. The cells are washed again with D-PBS three times. Nonspecificbinding sites on the microtiter plate are blocked with 2% bovine serumalbumin in D-PBS for 1 hour at 37° C. Cells are incubated with theappropriate monoclonal antibody diluted in blocking solution for 1 hourat 37° C. Unbound antibody is removed by washing the cells three timeswith D-PBS. Antibody bound to the cells is detected by incubation with a1:1000 dilution of biotinylated goat anti-mouse IgG (Bethesda ResearchLaboratories, Gaithersberg, Md.) in blocking solution for 1 hour at 37°C. Cells are washed three times with D-PBS and then incubated with a1:1000 dilution of streptavidin conjugated to β-galactosidase (BethesdaResearch Laboratories) for 1 hour at 37° C. The cells are washed threetimes with D-PBS for 5 minutes each. The amount of β-galactosidase boundto the specific monoclonal antibody is determined by developing theplate in a solution of 3.3 mM chlorophenolred-β-D-galactopyranoside, 50mM sodium phosphate, 1.5 mM MgCl₂ ; pH=7.2 for 2 to 15 minutes at 37° C.The concentration of the product is determined by measuring theabsorbance at 575 nm in an ELISA microtiter plate reader.

An example of the induction of ICAM-1 observed following stimulationwith either interleukin-1β or tumor necrosis factor α in several humancell lines is shown in FIG. 4. Cells were stimulated with increasingconcentrations of interleukin-1 or tumor necrosis factor for 15 hoursand processed as described above. ICAM-1 expression was determined byincubation with a 1:1000 dilution of the monoclonal antibody 84H10 (AmacInc., Westbrook, Me.). The cell lines used were passage 4 humanumbilical vein endothelial cells (HUVEC), a human epidermal carcinomacell line (A431), a human melanoma cell line (SK-MEL-2) and a human lungcarcinoma cell line (A549). ICAM-1 was induced on all the cell lines,however, tumor necrosis factor was more effective than interleukin-1 ininduction of ICAM-1 expression on the cell surface (FIG. 4).

Screening antisense oligonucleotides for inhibition of ICAM-1, VCAM-1 orELAM-1 expression is performed as described above with the exception ofpretreatment of cells with the oligonucleotides prior to challenge withthe cytokines. An example of antisense oligonucleotide inhibition ofICAM-1 expression is shown in FIG. 5. Human umbilical vein endothelialcells (HUVEC) were treated with increasing concentration ofoligonucleotide diluted in Opti MEM (GIBCO, Grand Island, N.Y.)containing 8 μM N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammoniumchloride (DOTMA) for 4 hours at 37° C. to enhance uptake of theoligonucleotides. The medium was removed and replaced with endothelialgrowth medium (EGM-UV; Clonetics, San Diego, Calif.) containing theindicated concentration of oligonucleotide for an additional 4 hours.Interleukin-1β was added to the cells at a concentration of 5 units/mland incubated for 14 hours at 37° C. The cells were quantitated forICAM-1 expression using a 1:1000 dilution of the monoclonal antibody84H10 as described above. The oligonucleotides used were:

COMPOUND 1--(ISIS 1558) a phosphodiester oligonucleotide designed tohybridize with position 64-80 of the mRNA covering the AUG initiation oftranslation codon having the sequence

    ______________________________________                                        5'-TGGGAGCCATAGCGAGGC-3'                                                                          (SEQ ID NO: 1).                                           ______________________________________                                    

COMPOUND 2--(ISIS 1570) a phosphorothioate containing oligonucleoeidecorresponding to the same sequence as COMPOUND

COMPOUND 3--a phosphorothioate oligonucleotide complementary to COMPOUND1 and COMPOUND 2 exhibiting the sequence

5'-GCCTCGCTATGGCTCCCA-3' (SEQ ID NO: 81).

COMPOUND 4--(ISIS 1572) a phosphorothioate containing oligonucleotidedesigned to hybridize to positions 2190-2210 of the mRNA in the 3'untranslated region containing the sequence

5'-GACACTCAATAAATAGCTGGT-3' (SEQ ID NO: 3).

COMPOUND 5--(ISIS 1821) a phosphorothioate containing oligonucleotidedesigned to hybridize to human 5-lipoxygenase mRNA used as a controlcontaining the sequence

5'-CATGGCGCGGGCCGCGGG-3' (SEQ ID NO: 82).

The phosphodiester oligonucleotide targeting the AUG initiation oftranslation region of the human ICAM-1 mRNA (COMPOUND 1) did not inhibitexpression of ICAM-1; however, the corresponding phosphorothioatecontaining oligonucleotide (COMPOUND 2) inhibited ICAM-1 expression by70% at a concentration of 0.1 μM and 90% at 1 μM concentration (FIG. 4).The increased potency of the phosphorothioate oligonucleotide over thephosphodiester is probably due to increased stability. The sense strandto COMPOUND 2, COMPOUND 3, modestly inhibited ICAM-1 expression at 10μM. If COMPOUND 2 was prehybridized to COMPOUND 3 prior to addition tothe cells, the effects of COMPOUND 2 on ICAM-1 expression wereattenuated suggesting that the activity of COMPOUND 2 was due toantisense oligonucleotide effect, requiring hybridization to the mRNA.The antisense oligonucleotide directed against 3' untranslated sequences(COMPOUND 4) inhibited ICAM-1 expression 62% at a concentration of 1 μM(FIG. 5). The control oligonucleotide, targeting human 5-lipoxygenase(COMPOUND 5) reduced ICAM-1 expression by 20%. These data demonstratethat oligonucleotides are capable of inhibiting ICAM-1 expression onhuman umbilical vein endothelial cells and suggest that the inhibitionof ICAM-1 expression is due to an antisense activity.

The antisense oligonucleotide COMPOUND 2 at a concentration of 1 μMinhibits expression of ICAM-1 on human umbilical vein endothelial cellsstimulated with increasing concentrations of tumor necrosis factor andinterleukin-1 (FIG. 6). These data demonstrate that the effects ofCOMPOUND 2 are not specific for interleukin-1 stimulation of cells.

Analogous assays can also be used to demonstrate inhibition of ELAM-1and VCAM-1 expression by antisense oligonucleotides.

Example 2

A second cellular assay which can be used to demonstrate the effects ofantisense oligonucleotides on ICAM-1, VCAM-1 or ELAM-1 expression is acell adherence assay. Target cells are grown as a monolayer in amultiwell plate, treated with oligonucleotide followed by cytokine. Theadhering cells are then added to the monolayer cells and incubated for30 to 60 minutes at 37° C. and washed to remove nonadhering cells. Cellsadhering to the monolayer may be determined either by directly countingthe adhering cells or prelabeling the cells with a radioisotope such as⁵¹ Cr and quantitating the radioactivity associated with the monolayeras described. Dustin and Springer, J. Cell Biol. 1988, 107, 321-331.Antisense oligonucleotides may target either ICAM-1, VCAM-1 or ELAM-1 inthe assay.

An example of the effects of antisense oligonucleotides targeting ICAM-1mRNA on the adherence of DMSO differentiated HL-60 cells to tumornecrosis factor treated human umbilical vein endothelial cells is shownin FIG. 7. Human umbilical vein endothelial cells were grown to 80%confluence in 12 well plates. The cells were treated with 2 μMoligonucleotide diluted in Opti-MEM containing 8 μM DOTMA for 4 hours at37° C. The medium was removed and replaced with fresh endothelial cellgrowth medium (EGM-UV) containing 2 μM of the indicated oligonucleotideand incubated 4 hours at 37° C. Tumor necrosis factor, 1 ng/ml, wasadded to cells as indicated and cells incubated for an additional 19hours. The cells were washed once with EGM-UV and 1.6×10⁵ HL-60 cellsdifferentiated for 4 days with 1.3% DMSO added. The cells were allowedto attach for 1 hour at 37° C. and gently washed 4 times with Dulbecco'sphosphate-buffered saline (D-PBS) warmed to 37° C. Adherent cells weredetached from the monolayer by addition of 0.25 ml of cold (40° C.)phosphate-buffered saline containing 5 mM EDTA and incubated on ice for5 minutes. The number of cells removed by treatment with EDTA wasdetermined by counting with a hemocytometer. Endothelial cells detachedfrom the monolayer by EDTA treatment could easily be distinguished fromHL-60 cells by morphological differences.

In the absence of tumor necrosis factor, 3% of the HL-60 cells bound tothe endothelial cells. Treatment of the endothelial cell monolayer with1 ng/ml tumor necrosis factor increased the number of adhering cells to59% of total cells added (FIG. 7). Treatment with the antisenseoligonucleotide COMPOUND 2 or the control oligonucleotide COMPOUND 5 didnot change the number of cells adhering to the monolayer in the absenceof tumor necrosis factor treatment (FIG. 7). The antisenseoligonucleotide, COMPOUND 2 reduced the number of adhering cells from59% of total cells added to 17% of the total cells added (FIG. 7). Incontrast, the control oligonucleotide COMPOUND 5 did not significantlyreduce the number of cells adhering to the tumor necrosis factor treatedendothelial monolayer, i.e., 53% of total cells added for COMPOUND 5treated cells versus 59% for control cells.

These data indicate that antisense oligonucleotides are capable ofinhibiting ICAM-1 expression on endothelial cells and that inhibition ofICAM-1 expression correlates with a decrease in the adherence of aneutrophil-like cell to the endothelial monolayer in a sequence specificfashion. Because other molecules also mediate adherence of white bloodcells to endothelial cells, such as ELAM-1, and V(CAM-1 it is notexpected that adherence would be completely blocked.

Example 3 Synthesis and Characterization of Oligonucleotides

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

2'-O-methyl phosphorothioate oligonucleotides were synthesized using2'-O-methyl β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, NeedhamMass.) and the standard cycle for unmodified oligonucleotides, exceptthe wait step after pulse delivery of tetrazole and base was increasedto 360 seconds. The 3'-base used to start the synthesis was a2'-deoxyribonucleotide.

2'-fluoro phosphorothioate oligonucleotides were synthesized using5'-dimethoxytrityl-3'-phosphoramidites and prepared as disclosed in U.S.patent application Ser. Nos. 463,358, filed Jan. 11, 1990, and 566,977,filed Aug. 13, 1990, which are assigned to the same assignee as theinstant application and which are incorporated by reference herein. The2'-fluoro oligonucleotides were prepared using phosphoramidite chemistryand a slight modification of the standard DNA synthesis protocol:deprotection was effected using methanolic ammonia at room temperature.

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20%. acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and phosphorothioateoligonucleotides were judged from electrophoresis to be greater than 80%full length material.

RNA oligonucleotide synthesis was performed on an ABI model 380B DNAsynthesizer. The standard synthesis cycle was modified by increasing thewait step after the pulse delivery of tetrazole to 900 seconds. Thebases were deprotected by incubation in methanolic ammonia overnight.Following base deprotections the oligonucleotides were dried in vacuo.The t-butyldimethylsilyl protecting the 2' hydroxyl was removed byincubating the oligonucleotide in 1 M tetrabutylammonium-fluoride intetrahydrofuran overnight. The RNA oligonucleotides were furtherpurified on C₁₈ Sep-Pak cartridges (Waters, Division of Millipore Corp.,Milford Mass.) and ethanol precipitated.

The relative amounts of phosphorothioate and phosphodiester linkagesobtained by this synthesis were periodically checked by ³¹ P NMRspectroscopy. The spectra were obtained at ambient temperature usingdeuterium oxide or dimethyl sulfoxide-d₆ as solvent. Phosphorothioatesamples typically contained less than one percent of phosphodiesterlinkages.

Secondary evaluation was performed with oligonucleotides purified bytrityl-on HPLC on a PRP-1 column (Hamilton Co., Reno, Nev.) using agradient of acetonitrile in 50 mM triethylammonium acetate, pH 7.0 (4%to 32% in 30 minutes, flow rate=1.5 ml/min). Appropriate fractions werepooled, evaporated and treated with 5% acetic acid at ambienttemperature for 15 minutes. The solution was extracted with an equalvolume of ethyl acetate, neutralized with ammonium hydroxide, frozen andlyophilized. HPLC-purified oligonucleotides were not significantlydifferent in potency from precipitated oligonucleotides, as judged bythe ELISA assay for ICAM-1 expression.

Example 4 Cell Culture and Treatment with Oligonucleotides

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Bethesda Md.). Cells were grown in Dulbecco'sModified Eagle's Medium (Irvine Scientific, Irvine Calif.) containing 1gm glucose/liter and 10% fetal calf serum (Irvine Scientific). Humanumbilical vein endothelial cells (HUVEC) (Clonetics, San Diego Calif.)were cultured in EGM-UV medium (Clonetics). HUVEC were used between thesecond and sixth passages. Human epidermal carcinoma A431 cells wereobtained from the American Type Culture Collection and cultured in DMEMwith 4.5 g/l glucose. Primary human keratinocytes were obtained fromClonetics and grown in KGM (Keratinocyte growth medium, Clonetics).

Cells grown in 96-well plates were washed three times with Opti-MEM(GIBCO, Grand Island, N.Y.) prewarmed to 37° C. 100 μl of Opti-MEMcontaining either 10 μg/mlN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA,Bethesda Research Labs, Bethesda, Md.) in the case of HUVEC cells or 20μg/ml DOTMA in the case of A549 cells was added to each well.Oligonucleotides were sterilized by centrifugation through 0.2 μmCentrex cellulose acetate filters (Schleicher and Schuell, Keene, N.H.).Oligonucleotides were added as 20×stock solution to the wells andincubated for 4 hours at 37° C. Medium was removed and replaced with 150μl of the appropriate growth medium containing the indicatedconcentration of oligonucleotide. Cells were incubated for an additional3 to 4 hours at 37° C. then stimulated with the appropriate cytokine for14 to 16 hours, as indicated. ICAM-1 expression was determined asdescribed in Example 1. The presence of DOTMA during the first 4 hoursincubation with oligonucleotide increased the potency of theoligonucleotides at least 100-fold. This increase in potency correlatedwith an increase in cell uptake of the oligonucleotide.

Example 5 ELISA Screening of Additional Antisense Oligonucleotides forActivity against ICAM-1 Gene Expression in Interleukin-1β-StimulatedCells

Antisense oligonucleotides were originally designed that would hybridizeto five target sites on the human ICAM-1 mRNA. Oligonucleotides weresynthesized in both phosphodiester (P=O; ISIS 1558, 1559, 1563, 1564 and1565) and phosphorothioate (P=S; ISIS 1570, 1571, 1572, 1573, and 1574)forms. The oligonucleotides are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        ANTISENSE OLIGONUCLEOTIDES                                                    WHICH TARGET HUMAN ICAM-1                                                     ISIS  SEQ                                                                     NO.   ID NO.  TARGET REGION      MODIFICATION                                 ______________________________________                                        1558  1       AUG Codon (64-81)  P = O                                        1559  2       5'-Untranslated (32-49)                                                                          P = O                                        1563  3       3'-Untranslated (2190-3010)                                                                      P = O                                        1564  4       3'-Untranslated (2849-2866)                                                                      P = O                                        1565  5       Coding Region (1378-1395)                                                                        P = O                                        1570  1       AUG Codon (64-81)  P = S                                        1571  2       5'-Untranslated (32-49)                                                                          P = S                                        1572  3       3'-Untranslated (2190-3010)                                                                      P = S                                        1573  4       3'-Untranslated (2849-2866)                                                                      P = S                                        1574  5       Coding Region (1378-1395)                                                                        P = S                                        1930  6       5'-Untranslated (1-20)                                                                           P = S                                        1931  7       AUG Codon (55-74)  P = S                                        1932  8       AUG Codon (72-91)  P = S                                        1933  9       Coding Region (111-130)                                                                          P = S                                        1934  10      Coding Region (351-370)                                                                          P = S                                        1935  11      Coding Region (889-908)                                                                          P = S                                        1936  12      Coding Region (1459-1468)                                                                        P = S                                        1937  13      Termination Codon (1651-1687)                                                                    P = S                                        1938  14      Termination Codon (1668-1687)                                                                    P = S                                        1939  15      3'-Untranslated (1952-1971)                                                                      P = S                                        1940  16      3'-Untranslated (2975-2994)                                                                      P = S                                        2149  17      AUG Codon (64-77)  P = S                                        2163  18      AUG Codon (64-75)  P = S                                        2164  19      AUG Codon (64-73)  P = S                                        2165  20      AUG Codon (66-80)  P = S                                        2173  21      AUG Codon (64-79)  P = S                                        2302  22      3'-Untranslated (2114-2133)                                                                      P = S                                        2303  23      3'-Untranslated (2039-2058)                                                                      P = S                                        2304  24      3'-Untranslated (1895-1914)                                                                      P = S                                        2305  25      3'-Untranslated (1935-1954)                                                                      P = S                                        2307  26      3'-Untranslated (1976-1995)                                                                      P = S                                        2634  1       AUG-Codon (64-81)  2'-fluoro                                                                     A, C & U;                                                                     P = S                                        2637  15      3'-Untranslated (1952-1971)                                                                      2'-fluoro                                                                     A, C & U;                                    2691  1       AUG Codon (64-81)  P = O, except                                                                 last 3                                                                        bases, P = S                                 2710  15      3'-Untranslated (1952-1971)                                                                      2'-O-                                                                         methyl; P = O                                2711  1       AUG Codon (64-81)  2'-O-                                                                         methyl; P = O                                2973  15      3'-Untranslated (1952-1971)                                                                      2'-O-                                                                         methyl; P = S                                2974  1       AUG Codon (64-81)  2'-O-                                                                         methyl; P = S                                3064  27      5'-CAP (32-51)     P = S; G & C                                                                  added as                                                                      spacer to                                                                     3'                                           3067  84      5'-CAP (32-51)     P = S                                        3222  84      5'-CAP (32-51)     2'-O-                                                                         methyl; P = O                                3224  84      5'-CAP (32-51)     2'-O-                                                                         methyl; P = S                                3581  85      3'-Untranslated (1959-1978)                                                                      P = S                                        ______________________________________                                    

Inhibition of ICAM-1 expression on the surface ofinterleukin-1β-stimulated cells by the oligonucleotides was determinedby ELISA assay as described in Example 1. The oligonucleotides weretested in two different cell lines. None of the phosphodiesteroligonucleotides inhibited ICAM-1 expression. This is probably due tothe rapid degradation of phosphodiester oligonucleotides in cells. Ofthe five phosphorothioate oligonucleotides, the most active was ISIS1570, which hybridizes to the AUG translation initiation codon. A2'-o-methyl phosphorothioate oligonucleotide, ISIS 2974, wasapproximately threefold less effective than ISIS 1570 in inhibitingICAM-1 expression in HUVEC and A549 cells. A 2'-fluoro oligonucleotide,ISIS 2634, was also less effective.

Based on the initial data obtained with the five original targets,additional oligonucleotides were designed which would hybridize with theICAM-1 mRNA. The antisense oligonucleotide (ISIS 3067) which hybridizesto the predicted transcription initiation site (5' cap site) wasapproximately as active in IL-1β-stimulated cells as the oligonucleotidethat hybridizes to the AUG codon (ISIS 1570), as shown in FIG. 8. ISIS1931 and 1932 hybridize 5' and 3', respectively, to the AUG translationinitiation codon. All three oligonucleotides that hybridize to the AUGregion inhibit ICAM-1 expression, though ISIS 1932 was slightly lessactive than ISIS 1570 and ISIS 1931. Oligonucleotides which hybridize tothe coding region of ICAM-1 mRNA (ISIS 1933, 1934, 1935, 1574 and 1936)exhibited weak activity. Oligonucleotides that hybridize to thetranslation termination codon (ISIS 1937 and 1938) exhibited moderateactivity.

Surprisingly, the most active antisense oligonucleotide was ISIS 1939, aphosphorothioate oligonucleotide targeted to a sequence in the3'-untranslated region of ICAM-1 mRNA (see Table 1). Otheroligonucleotides having the same sequence were tested, 2'-O-methyl (ISIS2973) and 2'-fluoro (ISIS 2637); however, they did not exhibit thislevel of activity. Oligonucleotides targeted to other 3' untranslatedsequences (ISIS 1572, 1573 and 1940) were also not as active asISIS-1939. In fact, ISIS 1940, targeted to the polyadenylation signal,did not inhibit ICAM-1 expression.

Because ISIS 1939 proved unexpectedly to exhibit the greatest antisenseactivity of the original 16 oligonucleotides tested, otheroligonucleotides were designed to hybridize to sequences in the3'-untranslated region of ICAM-1 mRNA (ISIS 2302, 2303, 2304, 2305, and2307, as shown in Table 1). ISIS 2307, which hybridizes to a site onlyfive bases 3' to the ISIS 1939 target, was the least active of theseries (FIG. 8). ISIS 2302, which hybridizes to the ICAM-1 mRNA at aposition 143 bases 3' to the ISIS 1939 target, was the most active ofthe series, with activity comparable to that of ISIS 1939. Examinationof the predicted RNA secondary structure of the human ICAM-1 mRNA3'-untranslated region (according to M. Zuker, Science 1989, 244, 48-52)revealed that both ISIS 1939 and ISIS 2302 hybridize to sequencespredicted to be in a stable stem-loop structure. Current dogma suggeststhat regions of RNA secondary structure should be avoided when designingantisense oligonucleotides. Thus, ISIS 1939 and ISIS 2302 would not havebeen predicted to inhibit ICAM-1 expression.

The control oligonucleotide ISIS 1821 did inhibit ICAM-1 expression inHUVEC cells with activity comparable to that of ISIS 1934; however, inA549 cells ISIS 1821 was less effective than ISIS 1934. The negativecontrol, ISIS 1821, was found to have a small amount of activity againstICAM expression, probably due in part to its ability to hybridize (12 of13 base match) to the ICAM-1 mRNA at a position 15 bases 3' to the AUGtranslation initiation codon.

These studies indicate that the AUG translation initiation codon andspecific 3'-untranslated sequences in the ICAM-1 mRNA were the mostsusceptible to antisense oligonucleotide inhibition of ICAM-1expression.

In addition to inhibiting ICAM-1 expression in human umbilical veincells and the human lung carcinoma cells (A549), ISIS 1570, ISIS 1939and ISIS 2302 were shown to inhibit ICAM-1 expression in the humanepidermal carcinoma A431 cells and in primary human keratinocytes (shownin FIG. 9). These data demonstrate that antisense oligonucleotides arecapable of inhibiting ICAM-1 expression in several human cell lines.Furthermore, the rank order potency of the oligonucleotides is the samein the four cell lines examined. The fact that ICAM-1 expression couldbe inhibited in primary human keratinocytes is important becauseepidermal keratinocytes are an intended target of the antisensenucleotides.

Example 6 Antisense Oligonucleotide Inhibition of ICAM-1 Expression inCells Stimulated with other Cytokines

Two oligonucleotides, ISIS 1570 and ISIS 1939, were tested for theirability to inhibit TNF-α and IFN-γ-induced ICAM-1 expression. Treatmentof A549 cells with 1 μM antisense oligonucleotide inhibited IL-1β, TNF-αand IFN-γ-induced ICAM-1 expression in a sequence-specific manner. Theantisense oligonucleotides inhibited IL-1β and TNF-α-induced ICAM-1expression to a similar extent, while IFN-γ-induced ICAM-1 expressionwas more sensitive to antisense inhibition. The control oligonucleotide,ISIS 2821, did not significantly inhibit IL-1β- or TNF-α-induced ICAM-1expression and inhibited IFN-γ-induced ICAM-1 expression slightly, asfollows:

    ______________________________________                                        Antisense Oligonucleotide                                                     (% Control Expression)                                                        Cytokine   ISIS 1570   ISIS 1939                                                                              ISIS 1821                                     ______________________________________                                        3 U/ml IL-1β                                                                        56.6 ± 2.9                                                                             38.1 ± 3.2                                                                            95 ± 6.6                                 1 ng/ml TNF-α                                                                      58.1 ± 0.9                                                                             37.6 ± 4.1                                                                          103.5 ± 8.2                                100 U/ml   38.9 ± 3.0                                                                             18.3 ± 7.0                                                                           83.0 ± 3.5                                gamma-IFN                                                                     ______________________________________                                    

Example 7 Antisense Effects are Abolished by Sense Strand Controls

The antisense oligonucleotide inhibition of ICAM-1 expression by theoligonucleotides ISIS 1570 and ISIS 1939 could be reversed byhybridization of the oligonucleotides with their respective sensestrands. The phosphorothioate sense strand for ISIS 1570 (ISIS 1575),when applied alone, slightly enhanced IL-1β-induced ICAM-1 expression.Premixing ISIS 1570 with ISIS 1575 at equal molar concentrations, priorto addition to the cells, blocked the effects of ISIS 1570. Thecomplement to ISIS 1939 (ISIS 2115) enhanced ICAM-1 expression by 46%when added to the cells alone. Prehybridization of ISIS 2115 to ISIS1939 completely blocked the inhibition of ICAM-1 expression by ISIS1939.

Example 8 Measurement of Oligonucleotide Tm (Dissociation Temperature ofOligonucleotide from Target)

To determine if the potency of the inhibition of ICAM-1 expression byantisense oligonucleotides was due to their affinity for their targetsites, thermodynamic measurements were made for each of theoligonucleotides. The antisense oligonucleotides (synthesized asphosphorothioates) were hybridized to their complementary DNA sequences(synthesized as phosphodiesters). Absorbance vs. temperature profileswere measured at 4 μM each strand oligonucleotide in 100 mM Na+, 10 mMphosphate, 0.1 mM EDTA, pH7.0. Tm's and free energies of duplexformation were obtained from fits of data to a two-state model withlinear sloping baselines (Petersheim, M. and D. H. Turner, Biochemistry1983, 22, 256-263). Results are averages of at least three experiments.

When the antisense oligonucleotides were hybridized to theircomplementary DNA sequences (synthesized as phosphodiesters), all of theantisense oligonucleotides with the exception of ISIS 1940 exhibited aTm of at least 50° C. All the oligonucleotides should therefore becapable of hybridizing to the target ICAM-1 mRNA if the target sequenceswere exposed. Surprisingly, the potency of the antisense oligonucleotidedid not correlate directly with either Tm or ΔG°₃₇. The oligonucleotidewith the greatest biological activity, ISIS 1939, exhibited a Tm whichwas lower than that of the majority of the other oligonucleotides. Thus,hybridization affinity is not sufficient to ensure biological activity.

Example 9 Effect of Oligonucleotide Length on Antisense Inhibition ofICAM-1 Expression

The effect of oligonucleotide length on antisense activity was testedusing truncated versions of ISIS 1570 (ISIS 2165, 2173, 2149, 2163 and2164) and ISIS 1939 (ISIS 2540, 2544, 2545, 2546, 2547 and 2548). Ingeneral, antisense activity decreased as the length of theoligonucleotides decreased. Oligonucleotides 16 bases in lengthexhibited activity slightly less than 18 base oligonucleotides.Oligonucleotides 14 bases in length exhibited significantly lessactivity, and oligonucleotides 12 or 10 bases in length exhibited onlyweak activity. Examination of the relationship between oligonucleotidelength and Tm and antisense activity reveals that a sharp transitionoccurs between 14 and 16 bases in length, while Tm increases linearlywith length (FIG. 10).

Example 10 Specificity of Antisense Inhibition of ICAM-1

The specificity of the antisense oligonucleotides ISIS 1570 and ISIS1939 for ICAM-1 was evaluated by immunoprecipitation of ³⁵ S-labelledproteins. A549 cells were grown to confluence in 25 cm² tissue cultureflasks and treated with antisense oligonucleotides as described inExample 4. The cells were stimulated with interleukin-1β for 14 hours,washed with methionine-free DMEM plus 10% dialyzed fetal calf serum, andincubated for 1 hour in methionine-free medium containing 10% dialyzedfetal calf serum, 1 μM oligonucleotide and interleukin-1β as indicated.³⁵ S-Methionine/cysteine mixture (Tran³⁵ S-label, purchased from ICN,Costa Mesa, Calif.) was added to the cells to an activity of 100 μCi/mland the cells were incubated an additional 2 hours. Cellular proteinswere extracted by incubation with 50 mM Tris-HCl pH 8.0, 150 mM NaCl,1.0% NP-40, 0.5% deoxycholate and 2 mM EDTA (0.5 ml per well) at 4° C.for 30 minutes. The extracts were clarified by centrifugation at18,000×g for 20 minutes. The supernatants were preadsorbed with 200 μlprotein G-Sepharose beads (Bethesda Research Labs, Bethesda Md.) for 2hours at 4° C., divided equally and incubated with either 5 μg ICAM-1monoclonal antibody (purchased from AMAC Inc., Westbrook Me.) or HLA-A,Bantibody (W6/32, produced by murine hybridoma cells obtained from theAmerican Type Culture Collection, Bethesda, Md.) for 15 hours at 4° C.Immune complexes were trapped by incubation with 200 μl of a 50%suspension of protein G-Sepharose (v/v) for 2 hours at 4° C., washed 5times with lysis buffer and resolved on an SDS-polyacrylamide gel.Proteins were detected by autoradiography.

Treatment of A549 cells with 5 units/ml of interleukin-1β was shown toresult in the synthesis of a 95-100 kDa protein migrating as a doubletwhich was immunoprecipitated with the monoclonal antibody to ICAM-1. Theappearance as a doublet is believed to be due to differentlyglycosylated forms of ICAM-1. Pretreatment of the cells with theantisense oligonucleotide ISIS 1570 at a concentration of 1 μM decreasedthe synthesis of ICAM-1 by approximately 50%, while 1 μM ISIS 1939decreased ICAM-1 synthesis to near background. Antisense oligonucleotideISIS 1940, inactive in the ICAM-1 ELISA assay (Examples 1 and 5) did notsignificantly reduce ICAM-1 synthesis. None of the antisenseoligonucleotides hybridizable with ICAM-1 targets had a demonstrableeffect on HLA-A, B synthesis, demonstrating the specificity of theoligonucleotides for ICAM-1. Furthermore, the proteins whichnonspecifically precipitated with the ICAM-1 antibody and proteinG-Sepharose were not significantly affected by treatment with theantisense oligonucleotides.

Example 11 Screening of Additional Antisense Oligonucleotides forActivity Against ICAM-1 by Cell Adhesion Assay

Human umbilical vein endothelial (HUVEC) cells were grown and treatedwith oligonucleotides as in Example 4. Cells were treated with eitherISIS 1939, ISIS 1940, or the control oligonucleotide ISIS 1821 for 4hours, then stimulated with TNF-α for 20 hours. Basal HUVEC minimallybound HL-60 cells, while TNF-stimulated HUVEC bound 19% of the totalcells added. Pretreatment of the HUVEC monolayer with 0.3 μM ISIS 1939reduced the adherence of HL-60 cells to basal levels, as shown in FIG.11. The control oligonucleotide, ISIS 1821, and ISIS 1940 reduced thepercentage of cells adhering from 19% to 9%. These data indicate thatantisense oligonucleotides targeting ICAM-1 may specifically decreaseadherence of a leukocyte-like cell line (HL-60) to TNF-α-treated HUVEC.

Example 12 ELISA Screening of Antisense Oligonucleotides for ActivityAgainst ELAM-1 Gene Expression

Primary human umbilical vein endothelial (HUVEC) cells, passage 2 to 5,were plated in 96-well plates and allowed to reach confluence. Cellswere washed three times with Opti-MEM (GIECO, Grand Island N.Y.). Cellswere treated with increasing concentrations of oligonucleotide dilutedin Opti-MEM containing 10 μg/ml DOTMA solution (Bethesda Research Labs,Bethesda Md.) for 4 hours at 37° C. The medium was removed and replacedwith EGM-UV (Clonetics, San Diego Calif.) plus oligonucleotide. Tumornecrosis factor α was added to the medium (2.5 ng/ml) and the cells wereincubated an additional 4 hours at 37° C.

ELAM-1 expression was determined by ELISA. Cells were gently washedthree times with Dulbecco's phosphate-buffered saline (D-PBS) prewarmedto 37° C. Cells were fixed with 95% ethanol at 4° C. for 20 minutes,washed three times with D-PBS and blocked with 2% BSA in D-PBS. Cellswere incubated with ELAM-1 monoclonal antibody BBA-1 (R&D Systems,Minneapolis Minn.) diluted to 0.5 μg/ml in D-PBS containing 2% BSA for 1hour at 37° C. Cells were washed three times with D-PBS and the boundELAM-1 antibody detected with biotinylated goat anti-mouse secondaryantibody followed by β-galactosidase-conjugated streptavidin asdescribed in Example 1.

The activity of antisense phosphorothioate oligonucleotides which target11 different regions on the ELAM-1 cDNA and two oligonucleotides whichtarget ICAM-1 (as controls) was determined using the ELAM-1 ELISA. Theoligonucleotide and targets are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        ANTISENSE OLIGONUCLEOTIDES                                                    WHICH TARGET HUMAN ELAM-1                                                     ISIS                                                                          NO.   SEQ ID NO.                                                                              TARGET REGION    MODIFICATION                                 ______________________________________                                        1926  28        AUG Codon (143-164)                                                                            P = S                                        2670  29        3'-Untranslated (3718-3737)                                                                    P = S                                        2673  30        3'-Untranslated (2657-2677)                                                                    P = S                                        2674  31        3'-Untranslated (2617-2637)                                                                    P = S                                        2678  32        3'-Untranslated (3558-3577)                                                                    P = S                                        2679  33        5'-Untranslated (41-60)                                                                        P = S                                        2680  34        3'-Untranslated (3715-3729)                                                                    P = S                                        2683  35        AUG Codon (143-163)                                                                            P = S                                        2686  36        AUG Codon (149-169)                                                                            P = S                                        2687  37        5'-Untranslated (18-37)                                                                        P = S                                        2693  38        3'-Untranslated (2760-2788)                                                                    P = S                                        2694  39        3'-Untranslated (2934-2954)                                                                    P = S                                        ______________________________________                                    

In contrast to what was observed with antisense oligonucleotidestargeted to ICAM-1 (Example 5), the most potent oligonucleotidemodulator of ELAM-1 activity (ISIS 2679) was hybridizable with specificsequences in the 5'-untranslated region of ELAM-1. ISIS 2687, anoligonucleotide which hybridized to sequences ending three basesupstream of the ISIS 2679 target, did not show significant activity(FIG. 12). Therefore, ISIS 2679 hybridizes to a unique site on theELAM-1 mRNA, which is uniquely sensitive to inhibition with antisenseoligonucleotides. The sensitivity of this site to inhibition withantisense oligonucleotides was not predictable based upon RNA secondarystructure predictions or information in the literature.

Example 13 ELISA Screening of Additional Antisense Oligonucleotides forActivity Against ELAM-1 Gene Expression

Inhibition of ELAM-1 expression by eighteen antisense phosphorothioateoligonucleotides was determined using the ELISA assay as described inExample 12. The target sites of these oligonucleotides on the ELAM-1mRNA are shown in FIG. 13. The sequence and activity of eacholigonucleotide against ELAM-1 are shown in Table 3. Theoligonucleotides indicated by an asterisk (*) have IC50's ofapproximately 50 nM or below and are preferred. IC50 indicates thedosage of oligonucleotide which results in 50% inhibition of ELAM-1expression.

                                      TABLE 3                                     __________________________________________________________________________    Inhibition of human ELAM-1 expression by antisense oligonucleotides           ELAM-1 expression is given as % of control                                    SEQ                               VCAM-1 EXPRESSION                           ISIS#                                                                             ID NO:                                                                            POSITION   SEQUENCE       30 nM oligo                                                                         50 nM oligo                           __________________________________________________________________________    *4764                                                                             52  5'-UTR                                                                             1-19  GAAGTCAGCCAAGAACAGCT                                                                         70.2  50.2                                   2687                                                                             37  5'-UTR                                                                             17-36 TATAGGAGTTTTGATGTGAA                                                                         91.1  73.8                                  *2679                                                                             33  5'-UTR                                                                             40-59 CTGCTGCCTCTGTCTCAGGT                                                                         6.4   6.0                                   *4759                                                                             53  5'-UTR                                                                             64-83 ACAGGATCTCTCAGGTGGGT                                                                         30.0  20.2                                  *2683                                                                             35  AUG  143-163                                                                             AATCATGACTTCAAGAGTTCT                                                                        47.9  48.5                                  *2686                                                                             36  AUG  148-168                                                                             TGAAGCAATCATGACTTCAAG                                                                        51.1  46.9                                  *4756                                                                             54  I/E  177-196                                                                             CCAAAGTGAGAGCTGAGAGA                                                                         53.9  35.7                                   4732                                                                             55  Coding                                                                             1936-1955                                                                           CTGATTCAAGGCTTTGGCAG                                                                         68.5  55.3                                  *4730                                                                             56  I/E 3'UTR                                                                          2006-2025                                                                           TTCCCCAGATGCACCTGTTT                                                                         14.1  2.3                                   *4729                                                                             57  3'-UTR                                                                             2063-2082                                                                           GGGCCAGAGACCCGAGGAGA                                                                         49.4  46.3                                  *2674                                                                             31  3'-UTR                                                                             2617-2637                                                                           CACAATCCTTAAGAACTCTTT                                                                        33.5  28.1                                   2673                                                                             30  3'-UTR                                                                             2656-2676                                                                           GTATGGAAGATTATAATATAT                                                                        58.9  53.8                                   2694                                                                             39  3'-UTR                                                                             2933-2953                                                                           GACAATATACAAACCTTCCAT                                                                        72.0  64.6                                  *4719                                                                             58  3'-UTR                                                                             2993-3012                                                                           ACGTTTGGCCTCATGGAAGT                                                                         36.8  34.7                                   4720                                                                             59  3'-UTR                                                                             3093-3112                                                                           GGAATGCAAAGCACATCCAT                                                                         63.5  70.6                                  *2678                                                                             32  3'-UTR                                                                             3557-3576                                                                           ACCTCTGCTGTTCTGATCCT                                                                         24.9  15.3                                   2670                                                                             29  3'-UTR                                                                             3717-3736                                                                           ACCACACTGGTATTTCACAC                                                                         72.2  67.2                                  __________________________________________________________________________     I/E indicates Intron/Exon junction                                            oligonucleotides with IC50's of approximately 50 nM or below are indicate     by an asterisk (*)                                                       

An additional oligonucleotide targeted to the 3'-untranslated region(ISIS 4728) did not inhibit ELAM expression.

Example 14 ELISA Screening of Antisense Oligonucleotides for ActivityAgainst VCAM-1 Gene Expression

Inhibition of VCAM-1 expression by fifteen antisense phosphorothioateoligonucleotides was determined using the ELISA assay approximately asdescribed in Example 12, except that cells were stimulated with TNF-αfor 16 hours and VCAM-1 expression was detected by a VCAM-1 specificmonoclonal antibody (R & D Systems, Minneapolis, Minn.) used at 0.5μg/ml. The target sites of these oligonucleotides on the VCAM-1 mRNA areshown in FIG. 14. The sequence and activity of each oligonucleotideagainst VCAM-1 are shown in Table 4. The oligonucleotides indicated byan asterisk (*) have IC50's of approximately 50 nM or below and arepreferred. IC50 indicates the dosage of oligonucleotide which results in50% inhibition of VCAM-1 expression.

                                      TABLE 4                                     __________________________________________________________________________    Inhibition of human VCAM-1 expression by antisense oligonucleotides           VCAM-1 expression is given as % of control                                    SEQ                                  VCAM-1 EXPRESSION                        ISIS#                                                                             ID NO:                                                                            POSITION      SEQUENCE       30 nM oligo                                                                         50 nM oligo                        __________________________________________________________________________    *5884                                                                             60  5'-UTR                                                                             1-19     CGATGCAGATACCGCGGAGT                                                                         79.2  37.2                                3791                                                                             61  5'-UTR                                                                             38-58    GCCTGGGAGGGTATTCAGCT                                                                         92.6  58.0                                5862                                                                             62  5'-UTR                                                                             48-68    CCTGTGTGTGCCTGGGAGGG                                                                         115.0 83.5                               *3792                                                                             63  AUG  110-129  GGCATTTTAAGTTGCTGTCG                                                                         68.7  33.7                                5863                                                                             64  CODING                                                                             745-764  CAGCCTGCCTTACTGTGGGC                                                                         95.8  66.7                               *5874                                                                             65  CODING                                                                             1032-1052                                                                              CTTGAACAATTAATTCCACCT                                                                        66.5  35.3                                5885                                                                             66  E/I  1633-1649 + intron                                                                     TTACCATTGACATAAAGTGTT                                                                        84.4  52.4                               *5876                                                                             67  CODING                                                                             2038-2057                                                                              CTGTGTCTCCTGTCTCCGCT                                                                         43.5  26.6                               *5875                                                                             68  CODING                                                                             2183-2203                                                                              GTCTTTGTTGTTTTCTCTTCC                                                                        59.2  34.8                                3794                                                                             69  TERMIN.                                                                            2344-2362                                                                              TGAACATATCAAGCATTAGC                                                                         75.3  52.6                               *3800                                                                             70  3'-UTR                                                                             2620-2639                                                                              GCAATCTTGCTATGGCATAA                                                                         64.4  47.7                               *3805                                                                             71  3'-UTR                                                                             2826-2845                                                                              CCCGGCATCTTTACAAAACC                                                                         67.7  44.9                               *3801                                                                             50  3'-UTR                                                                             2872-2892                                                                              AACCCAGTGCTCCCTTTGCT                                                                         36.5  21.3                               *5847                                                                             72  3'-UTR                                                                             2957-2976                                                                              AACATCTCCGTACCATGCCA                                                                         51.8  24.6                               *3804                                                                             51  3'-UTR                                                                             3005-3024                                                                              GGCCACATTGGGAAAGTTGC                                                                         55.1  29.3                               __________________________________________________________________________     E/I indicates excn/intron junction                                            Oligonucleotides with IC50's of approximately 50 nM or below are indicate     by an asterisk (*).                                                      

Example 15 ICAM-1 Expression in C8161 Human Melanoma Cells

Human melanoma cell Line C8161 (a gift of Dr. Dan Welch, Hershey MedicalCenter) was derived from an abdominal wall metastasis from a patientwith recurrent malignant melanoma. These cells form multiple metastasesin lung, subcutis, spleen, liver and regional lymph nodes aftersubcutaneous, intradermal and intravenous injection into athymic nudemice. Cells were grown in DMA-F12 medium containing 10% fetal calf serumand were passaged using 2 mM EDTA.

Exposure of C8161 cells to TNF-α resulted in a six-fold increase in cellsurface expression of ICAM-1 and an increase in ICAM-1 mRNA levels inthese cells. ICAM-1 expression on the cell surface was measured byELISA. Cells were treated with increasing concentrations of antisenseoligonucleotides in the presence of 15 μg/ml Lipofectin for 4 hours at37° C. ICAM-1 expression was induced by incubation with 5 ng/ml TNF-αfor 16 hours. Cells were washed 3× in DPBS and fixed for 20 minutes in2% formaldehyde. Cells were washed in DPBS, blocked with 2% BSA for 1hour at 37° C. and incubated with ICAM-1 monoclonal antibody 84H10(AMAC, Inc., Westbrooke, Me.). Detection of bound antibody wasdetermined by incubation with a biotinylated goat anti-mouse IgGfollowed by incubation with β-galactosidase-conjugated streptavidin anddeveloped with chlorophenol red-β-D-galactopyranoside and quantified byabsorbance at 575 nm. ICAM-1 mRNA levels were measured by Northern blotanalysis.

Example 16 Oligonucleotide Inhibition of ICAM-1 Expression in C8161Human Melanoma Cells

As shown in FIG. 15, antisense oligonucleotides ICAM 1570 (SEQ ID NO:1), ISIS 1939 (SEQ ID NO: 15) and ISIS 2302 (SEQ ID NO: 22) targeted toICAM-1 decreased cell surface expression of ICAM-1 (detected by ELISA asin Example 16). ISIS 1822, a negative control oligonucleotidecomplementary to 5-lipoxygenase, did not affect ICAM-1 expression. Thedata were expressed as percentage of control activity, calculated asfollows: (ICAM-1 expression for oligonucleotide-treated,cytokine-induced cells)--(basal ICAM-1 expression)/(ICAM-1cytokine-induced expression)--(basal ICAM-1 expression)×100.

ISIS 1939 (SEQ ID NO: 15) and ISIS 2302 (SEQ ID NO: 22) markedly reducedICAM-1 mRNA levels (detected by Northern blot analysis), but ISIS-1570(SEQ ID NO: 1) decreased ICAM-1 mRNA levels only slightly.

Example 17 Experimental Metastasis Assay

To evaluate the role of ICAM-1 in metastasis, experimental metastasisassays were performed by injecting 1×10⁵ C8161 cells into the lateraltail vein of athymic nude mice. Treatment of C8161 cells with thecytokine TNF-α and interferon γ has previously been shown to result inan increased number of lung metastases when cells were injected intonude mice [Miller, D. E. and Welch, D. R., Proc. Am. Assoc. Cancer Res.1990, 13, 353].

After 4 weeks, mice were sacrificed, organs were fixed in Bouin'sfixative and metastatic lesions on lungs were scored with the aid of adissecting microscope. Four-week-old female athymic nude mice (HarlanSprague Dawley) were used. Animals were maintained under the guidelinesof the NIH. Groups of 4-8 mice each were tested in experimentalmetastasis assays.

Example 18 Antisense Oligonucleotides ISIS 1570 and ISIS 2302 DecreaseMetastatic Potential of C8161 Cells

Treatment of C8161 cells with antisense oligonucleotides ISIS 1570 andISIS 2302, complementary to ICAM-1, was performed in the presence of thecationic lipid, Lipofectin (Gibco/BRL, Gaithersburg, Md.). Antisenseoligonucleotides were synthesized as described in Example 3. Cells wereseeded in 60 mm tissue culture dishes at 10⁶ cells/ml and incubated at37° C. for 3 days, washed with Opti-MEM (Gibco/BRL) 3 times and 100 μlof Opti-MEM medium was added to each well. 0.5 μM oligonucleotide and 15μg/ml lipofectin were mixed at room temperature for 15 minutes. 25 μl ofthe oligonucleotide-lipofectin mixture was added to the appropriatedishes and incubated at 37° C. for 4 hours. Theoligonucleotide-lipofectin mixture was removed and replaced with DME-F12medium containing 10% fetal calf serum. After 4 hours, 500 U/ml TNF-αwas added to the appropriate wells and incubated for 18 hours at whichtime cells were removed from the plates, counted and injected intoathymic nude mice.

Treatment of C8161 cells with ISIS 1570 (SEQ ID NO: 1) or ISIS 2302 (SEQID NO: 22) decreased the metastatic potential of these cells, andeliminated the enhanced metastatic ability of C8161 which resulted fromTNF-α treatment. Data are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Effect of antisense oligonucleotides to ICAM-1                                on experimental metastasis of human melanoma cell line C8161                                    No. Lung Metastases per Mouse                               Treatment         (Mean ± S.E.M.)                                          ______________________________________                                        Lipofectin only    64 ± 13                                                 Lipofectin + TNF-α                                                                        81 ± 8                                                   ISIS-1570 + Lipofectin                                                                           38 ± 15                                                 ISIS-2302 + Lipofectin                                                                          23 ± 6                                                   ISIS-1570 + Lipofectin + TNF-α                                                            49 ± 6                                                   ISIS-2302 + Lipofectin + TNF-α                                                            31 ± 8                                                   ______________________________________                                    

Example 19 Murine Models for Testing Antisense Oligonucleotides AgainstICAM-1

Many conditions which are believed to be mediated by intercellularadhesion molecules are not amenable to study in humans. For example,allograft rejection is a condition which is likely to be ameliorated byinterference with ICAM-1 expression, but clearly this must be evaluatedin animals rather than human transplant patients. Another such exampleis inflammatory bowel disease, and yet another is neutrophil migration(infiltration). These conditions can be tested in animal models,however, such as the mouse models used here.

Oligonucleotide sequences for inhibiting ICAM-1 expression in murinecells were identified. Murine ICAM-1 has approximately 50% homology withthe human ICAM-1 sequence; a series of oligonucleotides which target themouse ICAM-1 mRNA sequence were designed and synthesized, usinginformation gained from evaluation of oligonucleotides targeted to humanICAM-1. These oligonucleotides were screened for activity using animmunoprecipitation assay.

Murine DCEK-ICAM-1 cells (a gift from Dr. Adrienne Brian, University ofCalifornia at San Diego) were treated with 1 μM of oligonucleotide inthe presence of 20 μg/ml DOTMA/DOPE solution for 4 hours at 37° C. Themedium was replaced with methionine-free medium plus 10% dialyzed fetalcalf serum and 1 μmM antisense oligonucleotide. The cells were incubatedfor 1 hour in methionine-free medium, then 100 μCi/ml ³⁵ S-labeledmethionine/cysteine mixture was added to the cells. Cells were incubatedan additional 2 hours, washed 4 times with PBS, and extracted withbuffer containing 20 mM Tris, pH 7.2, 20 mM KCl, 5 mM EDTA, 1% TritonX-100, 0.1 mM leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. ICAM-1 wasimmunoprecipitated from the extracts by incubating with amurine-specific ICAM-1 antibody (YN1/1.7.4) followed by proteinG-sepharose. The immunoprecipitates were analyzed by SDS-PAGE andautoradiographed. Phosphorothioate oligonucleotides ISIS 3066 and 3069,which target the AUG codon of mouse ICAM-1, inhibited ICAM-1 synthesisby 48% and 63%, respectively, while oligonucleotides ISIS 3065 and ISIS3082, which target sequences in the 3'-untranslated region of murineICAM-1 mRNA inhibited ICAM-1 synthesis by 47% and 97%, respectively. Themost active antisense oligonucleotide against mouse ICAM-1 was targetedto the 3'-untranslated region. ISIS 3082 was evaluated further based onthese results; this 20-mer phosphorothioate oligonucleotide comprisesthe sequence (5' to 3') TGC ATC CCC CAG GCC ACC AT (SEQ ID NO: 83).

Example 20 Antisense Oligonucleotides to ICAM-1 Reduce InflammatoryBowel Disease in Murine Model System

A mouse model for inflammatory bowel disease (IBD) has recently beendeveloped. Okayasu et al., Gastroenterology 1990, 98, 694-702.Administration of dextran sulfate to mice induces colitis that mimicshuman IBD in almost every detail. Dextran sulfate-induced IBD and humanIBD have subsequently been closely compared at the histological leveland the mouse model has been found to be an extremely reproducible andreliable model. It is used here to test the effect of ISIS 3082, a20-base phosphorothioate antisense oligonucleotide which iscomplementary to the 3' untranslated region of the murine ICAM-1.

Female Swiss Webster mice (8 weeks of age) weighing approximately 25 to30 grams are kept under standard conditions. Mice are allowed toacclimate for at least 5 days before initiation of experimentalprocedures. Mice are given 5% dextran sulfate sodium in their drinkingwater (available ad libitum) for 5 days. Concomitantly, ISIS 3082oligonucleotide in pharmaceutical carrier, carrier alone (negativecontrol) or TGF-β (known to protect against dextran sulfate-mediatedcolitis in mice) is administered. ISIS 3082 was given as dailysubcutaneous injection of 1 mg/kg or 10 mg/kg for 5 days. TGF-β wasgiven as 1 μg/mouse intracolonically. At 1 mg/kg, the oligonucleotidewas as effective as TGF-β in protecting against dextran-sulfate-inducedcolitis.

Mice were sacrificed on day 6 and colons were subjected tohistopathologic evaluation. Until sacrifice, disease activity wasmonitored by observing mice for weight changes and by observing stoolsfor evidence of colitis. Mice were weighed daily. Stools were observeddaily for changes in consistency and for presence of occult or grossbleeding. A scoring system was used to develop a disease activity indexby which weight loss, stool consistency and presence of bleeding weregraded on a scale of 0 to 3 (0 being normal and 3 being most severelyaffected) and an index was calculated. Drug-induced changes in thedisease activity index were analyzed statistically. The disease activityindex has been shown to correlate extremely well with IRD in general.Results are shown in FIG. 16. At 1 mg/kg, the oligonucleotide reducedthe disease index by 40%.

Example 21 Antisense Oligonucleotide to ICAM-1 Increases Survival inMurine Heterotopic Heart Transplant Model

To determine the therapeutic effects of ICAM-1 antisense oligonucleotidein preventing allograft rejection the murine ICAM-1 specificoligonucleotide ISIS 3082 was tested for activity in a murinevascularized heterotopic heart transplant model. Hearts from Balb/c micewere transplanted into the abdominal cavity of C3H mice as primaryvascularized grafts essentially as described by Isobe et al.,Circulation 1991, 84, 1246-1255. Oligonucleotides were administered bycontinuous intravenous administration via a 7-day Alzet pump. The meansurvival time for untreated mice was 9.2±0.8 days (8, 9, 9, 9, 10, 10days). Treatment of the mice for 7 days with 5 mg/kg ISIS 3082 increasedthe mean survival time to 14.3±4.6 days (11, 12, 13, 21 days).

Example 22 Antisense Oligonucleotide to ICAM-1 Decreases LeukocyteMigration

Leukocyte infiltration of tissues and organs is a major aspect of theinflammatory process and contributes to tissue damage resulting frominflammation. The effect of ISIS 3082 on leukocyte migration wasexamined using a mouse model in which carrageenan-soaked sponges wereimplanted subcutaneously. Carrageenan stimulates leukocyte migration andedema. Effect of oligonucleotide on leukocyte migration in inflammatoryexudates is evaluated by quantitation of leukocytes infiltrating theimplanted sponges. Following a four hour fast, 40 mice were assignedrandomly to eight groups each containing five mice. Each mouse wasanesthetized with Metofane® and a polyester sponge impregnated with 1 mlof a 20 mg/ml solution of carrageenan was implanted subcutaneously.Saline was administered intravenously to Group 1 at 10 ml/kg four hoursprior to sponge implantation and this served as the vehicle control.Indomethacin (positive control) was administered orally at 3 mg/kg at avolume of 20 ml/kg to Group 2 immediately following surgery, again 6-8hours later and again at 21 hours post-implantation. ISIS 3082 wasadministered intravenously at 5 mg/kg to Group 3 four hours prior tosponge implantation. ISIS 3082 was administered intravenously at 5 mg/kgto Group 4 immediately following sponge implantation. ISIS 3082 wasadministered intravenously at 5 mg/kg to Groups 5, 6, 7 and 8 at 2, 4, 8and 18 hours following sponge implantation, respectively. Twenty-fourhours after implantation, sponges were removed, immersed in EDTA andsaline (5 ml) and squeezed dry. Total numbers of leukocytes in spongeexudate mixtures were determined.

The oral administration of indomethacin at 3 mg/kg produced a 79%reduction in mean leukocyte count when compared to the vehicle controlgroup.

A 42% reduction in mean leukocyte count was observed following theadministration of ISIS 3082 at 5 mg/kg four hours prior to spongeimplantation (Group 3). A 476 reduction in mean leukocyte count wasobserved following the administration of ISIS 3082 at 5 mg/kgimmediately following sponge implantation (Group 4). All animalsappeared normal throughout the course of study.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 87                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:                           #  18              GC                                                         - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 2:                           # 20               ACTG                                                       - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 3:                           #21                CTGG T                                                     - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 4:                           #  18              GA                                                         - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 5:                           #  18              AG                                                         - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 6:                           # 20               TAGC                                                       - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 7:                           # 20               TTGC                                                       - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:                           # 20               CCAT                                                       - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 9:                           # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 10:                          # 20               TTGA                                                       - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 11:                          # 20               GCCT                                                       - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 12:                          # 20               CCTT                                                       - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 13:                          # 20               TGTG                                                       - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 14:                          # 20               TTC A                                                      - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 15:                          # 20               TCTC                                                       - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 16:                          # 20               AACT                                                       - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 14                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 17:                          #     14                                                                      - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 18:                          #       12                                                                    - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 19:                          #        10                                                                   - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 20:                          #    16                                                                       - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 21:                          #    16                                                                       - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 22:                          # 20               GTCA                                                       - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 23:                          # 20               CCTC                                                       - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 24:                          # 20               CTTG                                                       - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 25:                          # 20               TTAA                                                       - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 26:                          # 20               CTAT                                                       - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 27:                          #                 22CTC GA                                                    - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 28:                          #                 22GTT CT                                                    - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 29:                          # 20               ACAC                                                       - (2) INFORMATION FOR SEQ ID NO: 30:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 30:                          #21                TATA T                                                     - (2) INFORMATION FOR SEQ ID NO: 31:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 31:                          #21                TCTT T                                                     - (2) INFORMATION FOR SEQ ID NO: 32:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 32:                          # 20               TCCT                                                       - (2) INFORMATION FOR SEQ ID NO: 33:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 33:                          # 20               AGGT                                                       - (2) INFORMATION FOR SEQ ID NO: 34:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 34:                          #    15                                                                       - (2) INFORMATION FOR SEQ ID NO: 35:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 35:                          #21                GTTC T                                                     - (2) INFORMATION FOR SEQ ID NO: 36:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 36:                          #21                TCAA G                                                     - (2) INFORMATION FOR SEQ ID NO: 37:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 37:                          # 20               TGAA                                                       - (2) INFORMATION FOR SEQ ID NO: 38:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 38:                          #21                CTAC A                                                     - (2) INFORMATION FOR SEQ ID NO: 39:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 39:                          #21                TCCA T                                                     - (2) INFORMATION FOR SEQ ID NO: 40:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 40:                          #21                GCTG T                                                     - (2) INFORMATION FOR SEQ ID NO: 41:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 41:                          # 20               CCCG                                                       - (2) INFORMATION FOR SEQ ID NO: 42:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 42:                          #21                AACC A                                                     - (2) INFORMATION FOR SEQ ID NO: 43:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 43:                          # 19               GAT                                                        - (2) INFORMATION FOR SEQ ID NO: 44:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 44:                          # 19               TGT                                                        - (2) INFORMATION FOR SEQ ID NO: 45:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 45:                          #                 22TAG CT                                                    - (2) INFORMATION FOR SEQ ID NO: 46:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 46:                          #                 22TAT GT                                                    - (2) INFORMATION FOR SEQ ID NO: 47:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 47:                          #                 22ACA AA                                                    - (2) INFORMATION FOR SEQ ID NO: 48:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 48:                          #21                GTTG T                                                     - (2) INFORMATION FOR SEQ ID NO: 49:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 49:                          # 20               TTTA                                                       - (2) INFORMATION FOR SEQ ID NO: 50:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 50:                          # 20               TGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 51:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 51:                          # 20               TTGC                                                       - (2) INFORMATION FOR SEQ ID NO: 52:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 52:                          # 20               AGCT                                                       - (2) INFORMATION FOR SEQ ID NO:   53:                                        -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 53:                          # 20               GGGT                                                       - (2) INFORMATION FOR SEQ ID NO: 54:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 54:                          # 20               GAGA                                                       - (2) INFORMATION FOR SEQ ID NO: 55:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 55:                          # 20               GCAG                                                       - (2) INFORMATION FOR SEQ ID NO: 56:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 56:                          # 20               GTTT                                                       - (2) INFORMATION FOR SEQ ID NO: 57:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 57:                          # 20               GAGA                                                       - (2) INFORMATION FOR SEQ ID NO: 58:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 58:                          # 20               AAGT                                                       - (2) INFORMATION FOR SEQ ID NO: 59:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 59:                          # 20               CCAT                                                       - (2) INFORMATION FOR SEQ ID NO: 60:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 60:                          # 20               GAGT                                                       - (2) INFORMATION FOR SEQ ID NO: 61:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 61:                          # 20               AGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 62:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 62:                          # 20               AGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 63:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 63:                          # 20               GTCG                                                       - (2) INFORMATION FOR SEQ ID NO: 64:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 64:                          # 20               GGGC                                                       - (2) INFORMATION FOR SEQ ID NO: 65:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   21                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 65:                          #21                CACC T                                                     - (2) INFORMATION FOR SEQ ID NO: 66:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   21                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 66:                          #21                GTGT T                                                     - (2) INFORMATION FOR SEQ ID NO: 67:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 67:                          # 20               CGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 68:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   21                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 68:                          #21                CTTC C                                                     - (2) INFORMATION FOR SEQ ID NO: 69:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 69:                          # 20               TAGC                                                       - (2) INFORMATION FOR SEQ ID NO: 70:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 70:                          # 20               ATAA                                                       - (2) INFORMATION FOR SEQ ID NO: 71:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 71:                          # 20               AACC                                                       - (2) INFORMATION FOR SEQ ID NO: 72:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   20                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 72:                          # 20               GCCA                                                       - (2) INFORMATION FOR SEQ ID NO: 73:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  22                                                               (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 73:                          #                 22TCA GG                                                    - (2) INFORMATION FOR SEQ ID NO: 74:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 23                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 74:                          #                23AAAA GGA                                                   - (2) INFORMATION FOR SEQ ID NO: 75:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 75:                          # 20               GGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 76:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 76:                          # 19               GTC                                                        - (2) INFORMATION FOR SEQ ID NO: 77:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 77:                          # 20               TTCA                                                       - (2) INFORMATION FOR SEQ ID NO: 78:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 78:                          #21                TGAC T                                                     - (2) INFORMATION FOR SEQ ID NO: 79:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 79:                          #                 22AAC CT                                                    - (2) INFORMATION FOR SEQ ID NO: 80:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 80:                          #                 22GCA TT                                                    - (2) INFORMATION FOR SEQ ID NO: 81:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 81:                          #  18              CA                                                         - (2) INFORMATION FOR SEQ ID NO: 82:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 82:                          #  18              GG                                                         - (2) INFORMATION FOR SEQ ID NO: 83:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 83:                          # 20               CCAT                                                       - (2) INFORMATION FOR SEQ ID NO: 84:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 84:                          # 20               GCTC                                                       - (2) INFORMATION FOR SEQ ID NO: 85:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 85:                          # 20               CTTC                                                       - (2) INFORMATION FOR SEQ ID NO: 86:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 37                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 86:                          #      37          GGTA TCGCTCCGAC TCCAACG                                    - (2) INFORMATION FOR SEQ ID NO: 87:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 8                                                                 (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 87:                          #           8                                                                 __________________________________________________________________________

What is claimed is:
 1. An antisense oligonucleotide targeted to atranslation initiation site of a nucleic acid presented in FIG. 1encoding intercellular adhesion molecule-1, said antisenseoligonucleotide comprising from 8 to 25 nucleotide subunits, whereinsaid antisense oligonucleotide modulates the production of saidintercellular adhesion molecule-1.
 2. The antisense oligonucleotide ofclaim 1 comprising from 12 to 22 nucleotide subunits.
 3. The antisenseoligonucleotide of claim 1, said antisense oligonucieotide having from12 to 22 nucleotide subunits comprising a sequence which hybridizes viaWatson-Crick base-pairing with a sequence contained within thesequence3'-GCCCCCGACGACCCTCGGTATCGCTCCGACTCCAACG-5' (SEQ ID NO:86)thereby modulating production of said intercellular adhesion molecule-1.4. An antisense oligonucleotide up to 50 nucleic acid base units inlength and comprising the sequence

    ______________________________________                                        5'-CGGGGGCTGCTGGGAGCCAT                                                                              SEQ ID NO:8,                                           5'-TGGGAGCCATAGCGAGGC  SEQ ID NO:1,                                           5'-TGGGAGCCATAGCGAG    SEQ ID NO:20,                                          5'-GGAGCCATAGCGAGGC    SEQ ID NO:21,                                          5'-AGCCATAGCGAGGC      SEQ ID NO:17,                                          5'-CCATAGCGAGGC        SEQ ID NO:18,                                          5'-CATAGCGAGGCTGAGGTTGC                                                                              SEQ ID NO:7, or                                        5'-ATAGCGAGGC          SEQ ID NO:19.                                          ______________________________________                                    